This invention relates to multiple stage mass spectrometers which have two mass analyzers, and this invention is more particularly concerned with both a method of and an apparatus for providing multiple stages of mass spectrometry (MSn) capabilities in such spectrometers.
Tandem mass spectrometry is widely used for trace analysis and for the determination of the structures of ions. In tandem mass spectrometry a first mass analyzer selects ions of one particular mass to charge ratio (or range of mass to charge ratios) from ions supplied by an ion source, the ions are fragmented and a second mass analyzer records the mass spectrum of the fragment ions. In a triple quadrupole mass spectrometer system, this effects MS/MS. For example, ions produced in an atmospheric pressure source, pass through a region of dry nitrogen and then pass through a small orifice, into a region at a pressure of about 5 torr (0.7 kPa). Ions then pass through a quadrupole ion guide, operated at a pressure of about 7xc3x9710xe2x88x923 torr (9.1xc3x9710xe2x88x924 kPa) into a first quadrupole mass filter, operated at a pressure of about 2xc3x9710xe2x88x925 torr (2.6xc3x9710xe2x88x926 kPa). Precursor ions mass selected in the first quadrupole are injected into a collision cell filled with gas, such as argon, to a pressure of 10xe2x88x924 to 10xe2x88x922 torr (1.3xc3x9710xe2x88x925 to 1.3xc3x9710xe2x88x923 kPa). The collision cell contains a second quadrupole ion guide, to confine ions to the axis. Ions gain internal energy through collisions with the gas and then fragment. The fragment ions and any undissociated precursor ions then pass into a second mass analyzer, and then to a detector, where the mass spectrum is recorded.
Triple quadrupole systems are widely used for tandem mass spectrometry. One limitation is that recording a fragment mass spectrum can be time consuming because the second mass analyzer must step through many masses to record a complete spectrum. To overcome these limitations, QqTOF systems have been developed. This system is similar to the triple quadrupole system but the second mass analyzer is replaced by a time-of-flight mass analyzer, TOF. The advantage of the TOF is that it can record 104 or more complete mass spectra in one second. Thus for applications where a complete mass spectrum of fragment ions is desired the duty cycle is greatly improved with a TOF mass analyzer and spectra can be acquired more quickly. Alternatively for a given measurement time, spectra can be acquired on a smaller amount of sample.
A further known technique is the coupling of electrospray ionization (ESI) to time-of-flight mass spectrometers (TOFMS), and this is an attractive technique for mass spectrometry. ESI is a soft ionization technique capable of forming ions from a broad range of biomolecules, while TOFMS has the well known advantages of rapid mass scanning, high sensitivity, and a theoretically limitless mass range. However, ESI and TOFMS are, in one way, incompatible as a source/analyzer pair: ESI creates a continuous stream of ions and TOFMS requires pulsed operation. Thus in the simplest coupling of ESI to TOFMS there is a very poor duty cycle, with less than 1% of the ions formed being detected and early work in this field was predominantly concerned with increasing the duty cycle.
Within the past two years, literature on ESI-TOFMS has begun to focus on tandem mass spectrometry (MS/MS) with hybrid instruments. The fragmentation of ions in these systems is achieved via traditional methods for collision induced dissociation (CID). Tandem-in-space systems termed quadrupole-TOF""s or xe2x80x9cQq-TOF""sxe2x80x9d, as noted above, are analogous to triple quadrupole mass spectrometersxe2x80x94the precursor ion is selected in a quadrupole mass filter, dissociated in a radiofrequency- (RF-) only multipole collision cell, and the resultant fragments are analyzed in a TOFMS. Tandem-in-time systems use a 3-D ion trap mass spectrometer (ITMS) for selecting and fragmenting the precursor ion, but pulse the fragment ions out of the trap and into a TOFMS for mass analysis.
It is sometimes desirable to perform multiple stages of tandem spectrometry termed MSn. In MS3 for example, a precursor ion is selected in a first mass analyzer and dissociated to produce fragment ions. A fragment ion of a particular mass to charge ratio is then isolated and dissociated again to produce fragments of the fragment. The mass spectrum of these is then recorded. Multiple stages of MS are useful when insufficient dissociation can be produced in a first stage of MS/MS or to elucidate dissociation pathways of complex ions. The latter for example is especially useful to sequence peptides and other biomolecules by mass spectrometry.
The triple quadrupole system and QqTOF system described above provide only one stage of MS/MS and do not allow MSn. In particular such systems do not provide for trapping of ions.
In one known proposal, in PCT application WO 98/06481 from Analytica of Brantford, there is a described system including ion trapping. Ions from a source are injected into a multipole ion guide, and ions of one m/z or range of m/z are then isolated in the ion guide by applying resonant excitation or AC/DC voltages to the ion guide and trapping voltages at either end. The ion is then fragmented in the same ion guide, which can be operated as a linear ion trap (LM. No mass analyzer is placed before the ion guide. This is a distinct disadvantage, since a multipole ion guide used both for ion isolation and mass analysis has a relatively low resolution. For example, the present inventors have found that using a LIT as described by Analytica the resolution in isolating an ion is ca. 100. With a separate quadrupole mass filter or other mass analyzer before the ion trap the resolution can be many thousand. The relatively low resolution for ions introduced into the multipole ion trap may derive from at least two sources: (1) the pressure is relatively high (10xe2x88x923-10xe2x88x921 torr (1.3xc3x9710xe2x88x924 to 1.3xc3x9710xe2x88x922 kPa) as described in the PCT application); and (2) in the system described in the PCT application the gas is either nitrogen or air that flows in from the ion source. This has a greater damping effect on ion motion in the LIT than lighter gases such as helium, and gives relatively poor resolution for resonant excitation of ions. Such a system does not readily enable the pressure and type of gas in the LIT to be adjusted to provide optimum conditions for MSn.
Additionally, there have been some recent examples of proposals using resonant excitation in RF-only quadrupoles for CID with fragment mass analysis by TOFMS. Dodonov et al (Rap. Comm. Mass Spectrometry 11, 1649-1656 (1997)) introduced a molecular ion reactor (MIR) consisting of a segmented RF-only quadrupole with a longitudinal electrical field which is operated at a high pressure. Depending on the mode of operation, CID was accomplished through either increasing the RF or DC voltages along the segments. However, no trapping of ions was demonstrated.
Loboda et al (proceedings of the 46th ASMS Conference on Mass Spectrometry and Allied Topics, Orlando, Fla., May 31-Jun. 4, 1998, MOD. 11: 55) modified the RF drive of the collision cell in a Q-TOFMS to apply quadrupolar excitation to ions flowing through the cell, inducing fragmentation. No trapping of ions was demonstrated. It was suggested that a 2D trap might be formed to isolate precursor ions, but it was not stated if this was to be done before or after a stage of mass analysis.
B. A. Thomson et al, in PCT application PCT/CA96/00541, describes a method and apparatus for speeding up the passage of ions through various stages of a mass spectrometer, such as the ion guide and the collision cell. The increase in ion speed is achieved via an axial DC field which can be created through various multipole rod configurations. The axial DC field also aids in the dissociation of ions in collision cells by oscillating the ions axially about their equilibrium positions. However, Thomson states that there is no need to operate at the resonant frequency of the ions or even at a harmonic of the resonant frequency of the ions.
J. D. Watson et al, in an article entitled xe2x80x9cA Technique for Mass Selective Ion Rejection in a Quadrupole Reaction Chamberxe2x80x9d (International Journal of Mass Spectrometry and Ion Processes, 93, 225-235, 1989) described trapping and resonant ejection of an ion from the quadrupole collision cell of a triple quadrupole mass spectrometer. The intent was to study reaction kinetics of trapped ions. While there is no specific teaching of resonant excitation of trapped ions without ejection, there is speculation that this might be possible.
In mass spectrometry, the linear ion trap has remained relatively unexplored. U.S. Pat. Nos. 4,755,670 and 5,420,425, both assigned to the Finnigan Corporation, relate to a Fourier transform quadrupole and new ion trap geometries respectively, and they both mention a LIT. U.S. Pat. No. 5,179,278 (D. J. Douglas) suggests using a LIT as an xe2x80x9cion bottlexe2x80x9d to improve the duty cycle of a 3D ITMS.
In accordance with a first aspect of the present invention, there is provided a method of analyzing a stream of ions, the method comprising:
(1) subjecting a stream of ions to a first mass analysis step, to select ions having a mass-to-charge ratio in a first desired range;
(2) passing ions in the selected range into a radio frequency linear ion trap containing gas;
(3) trapping the selected ions in the linear ion trap and exciting the ions to cause collisions with the ambient gas and fragmentation;
(4) subjecting the fragmented ions to a secondary excitation, different from the first excitation, to cause excitation and fragmentation of selected fragment ions; and
(5) passing the ions out of the linear ion trap and subjecting the ions to a further mass analysis step to determine the mass spectrum of the ions.
Passing the ions, in step (2) into the radio frequency ion trap can be done either: with a relatively low energy, so no fragmentation occurs in the LIT until additional excitation is applied; or with a relatively high energy in the axial direction, so that fragmentation occurs simply due to the high energy of the ions entering the LIT and colliding with the gas.
Thus a variant of the basic method of the present invention comprises passing the ions into the linear ion trap with sufficient energy to promote collision induced dissociation, said energy providing the excitation of (3), whereby step (3) comprises applying a signal to the linear ion trap to trap ions, before subjecting the ions to the further mass analysis of step (5).
In either case, once the ions have entered the LIT, have been excited, by either technique, to cause fragmentation, one then has fragmentions, with any remaining precursor ions trapped in the LIT. These ions can then be discharged for further mass analysis, or subject to multiple steps of mass selection and excitation to cause fragmentation, before being discharged for the final mass analysis step.
Thus, the method advantageously includes, in step (4), subjecting the fragmented ions to a secondary excitation, different from the first excitation, to cause excitation and fragmentation of selected fragment ions (MS3). This can be repeated to achieve further steps of MSn (n greater than 3). Further, prior to the additional step of secondary excitation, applying a signal to the linear ion trap, to select ions having a mass-to-charge ratio in a second desired range, wherein the secondary excitation step comprises exciting ions in the second desired range.
Thus, the method can include, while trapping the ions in the linear ion trap, effecting multiple cycles of:
(1) selecting ions having a mass-to-charge ratio in a desired range; and
(2) exciting the selected ions to cause fragmentation.
The ions can be excited in the linear ion trap by providing an additional signal to the linear ion trap.
The further mass analysis step of step (5) can be carried out either in a quadrupole mass analyzer, or in a time of flight mass analyzer. For a time of flight mass analyzer, this can be arranged with its axis perpendicular to the axis of the linear ion trap.
Preferably, the first mass analysis step is carried out in a quadrupole mass analyzer which is coaxial with the linear ion trap.
More preferably; the method includes, prior to exciting the ions in step (3), subjecting the trapped ions to a signal comprising a plurality of excitation signals uniformly spaced in the frequency domain and having a notch, wherein the notch covers a desired frequency band and there are no excitation signals in the frequency band of the notch, and wherein the excitation signals have sufficient magnitude to excite and eject ions except for ions having an excitation frequency falling within the frequency band of the notch. For the case where the frequency of the trapping RF signal is 1.0 MHz, this can be achieved by applying a combination of signals having sine waves with frequencies in the range 10 to 500 kHz and spaced at 500 Hz intervals, and the frequency band of the notch then has a width of typically 1-10 kHz and is centered on the resonant frequency of an ion of interest. More generally, where the trapping RF frequency is f, then the auxiliary frequencies should be up to f/2.
In accordance with another aspect of the present invention, there is provided an apparatus, for effecting mass analysis and fragmentation of an ion stream, the apparatus comprising:
an input for an ion stream;
a first mass analyzer;
a radio frequency linear ion trap; and
a final mass analyzer.
Preferably, the first mass analyzer comprises a quadrupole mass analyzer, and the final mass analyzer comprises a quadrupole mass analyzer, and the first mass analyzer, the linear ion trap and the final mass analyzer are axially aligned with one another.
The Radio frequency linear ion trap (LIT) could be formed in a number of ways. It could have aperture plates or lens at either end serving to provide the necessary D.C. potential gradient, to keep ions within the trap. Alternatively, where the ion trap is a multipole rod set, the rods can be segmented to permit different D.C. potentials to be applied to different segments. A segmented rods set also enables an axial D.C. field to be established.
For the final mass analyzer of the apparatus just defined and for the mass analysis step (4) of the method defined previously, the mass analyzer could be any suitable analyzer. Such an analyzer could be: a linear quadrupole, a linear or reflection TOF, a single magnetic sector analyzer; a double focusing two sector mass analyzer (having electric and magnetic sectors), a Paul trap (3D trap), a Wien filter, a Mattauch-Herzog spectrograph, a Thomson parabolic mass spectrometer, an ion cyclotron resonance mass spectrometer, etc.
The linear ion trap can be a multipole trap, but preferably includes a quadrupole rod set and the rods of the mass analyzers and of the linear ion trap preferably have substantially similar radii and substantially similar spacings.
The linear ion trap can have a pair of opposed x rods and a pair of opposed y rods, and then a main RF drive is connected to the x and y rods of the linear ion trap and an auxiliary drive is connected to at least one pair of rods of the linear ion trap. For example, the auxiliary drive is connected between the x and the y rods of the linear ion trap through a transformer, and the main RF drive is connected directly to the x rods of the linear ion trap and, through a coil of the transformer to the y rods. Alternatively, the auxiliary drive can be connected between the x rods. The apparatus preferably then includes an arbitrary waveform generator connected to the auxiliary drive, for applying a selected waveform to the linear ion trap to excite ions therein.